The rat prolactin gene (PRL) provides an useful model system to examine a number of aspects of gene regulation: 1) Activation and repression of expression; expression of PRL is regulated by both positive acting factors (e.q. estrogen receptor) and negative acting factors (e.q. glucocorticoid receptor); 2) Cell-specific expression; PRL is only expressed in lactotroph cells of the anterior pituitary; and 3) Action at a distance; both hormone and cell-specific expression is mediated through two distinct transcriptional regulatory elements separated by 1500 bp within the PRL gene's 5' flanking DNA sequences. Much of our current understanding of PRL gene regulation has come from transient transfection studies or in vitro protein-DNA binding studies. However, these studies do not take into account the complexities of the PRL gene structure as it exist in the cell nucleus. In vivo, PRL DNA is not simply a series of nucleotide sequences that are readily available for interaction with specific protein mediators of transcription. In vivo, PRL DNA is supercoiled and organized into a nucleosome-bound chromatin structure. This project focuses on understanding the effect of chromatin structure on the mechanism of steroid hormone regulation of PRL gene expression. We have developed a unique amplified minichromosome system that contains the transcriptional regulatory elements of PRL. The PRL-minichromosome responds in a manner similar to the endogenous PRL gene with respect to transcriptional regulation and alterations in its highly ordered chromatin structure. The PRLminichromosome system will be used to examine at the nucleotide level changes in protein binding to the PRL chromatin in response to steroid hormones and cell-specific factors that regulate transcription. These studies will identify specific regions of the PRL chromatin where proteins bind to the PRL DNA and possibly interact with each other during various stages of PRL expression. We intend to isolate these proteins, characterize their chromatin binding properties, and through the use of an in vitro transcription assay using isolated PRL-minichromosomes as templates, determine the role of these proteins in regulating the transcription of chromatin-associated PRL. Finally, the interaction between proteins bound to the two distinct regulatory regions in PRL chromatin and their role in PRL expression will be examined using a novel nuclear ligation assay and in vivo crosslinking studies.